Macromolecular assembly is a central but still poorly understood aspect of biological growth and development. Complex bacteriophages provide excellent model systems for investigation of this process. Studies in our laboratoory and others over the past ten years have shown that bacteriophage morphogenesis proceeds by strictly ordered assembly pathways, some of which include steps that require nonstructural accessory proteins to promote noncovalent associations of structural components. Basic understanding of macromolecular assembly will require elucidation of these phenomena, which cannot be explained by classical notions of self-assembly. We plan to exploit the advantages of bacteriophage T4 for genetic, biochemical, and ultrastructural analysis to investigate the mechansims of both sequential ordering and accessory protein action. Using T4 tail assembly as a model, we shall explore alternative explanations for how the temporal order of association steps in a self-assembly pathway can be dictated by the structural proteins themselves. In continiuing work on T4 tail fiber assembly and attachment, we shall investigate the mechanisms of three accessory proteins that appear to promote noncovalent associations of structural components. We plan to test the suppositon that these accessory proteins act to overcome kinetic barriers to association, to understand the nature of these barriers, and to determine how they are overcome. The assembly of bacteriophage capsids and the packaging of viral DNA also pose major unanswered questions that are applicable to many viruses. The answers could provide not only basic information on viral morphogenesis but also potentially valuable methods for interfering selectively with viral multiplication. We shall continue to investigate the roles of T4 RNA ligase, which we have shown to be involved somehow in capsid assembly and DNA packaging. In addition we shall exploit bacteriophage T7, which unlike T4 can be made to package its DNA efficiently in cell-free extracts, to better define the requirements of this process and to investigate the action of packaging inhibitors.